Natural Killer Cells (or NK cells) are a type of cytotoxic lymphocyte critical to the innate immune system. The role NK cells play is analogous to that of cytotoxic T cells in the vertebrate adaptive immune response. NK cells provide rapid responses to virally infected cells and respond to tumor formation, acting at around 3 days after injection. Typically immune cells detect MHC (major histocompatibility complex) presented on infected cell surfaces, triggering cytokine release, causing lysis or apoptosis. NK cells are unique, however, as they have the ability to recognize stressed cells in the absence of antibodies and MHC, allowing for a much faster immune reaction. They were named “natural killers” because of the initial notion that they do not require activation in order to kill cells that are missing “self” markers of major histocompatibility complex (MHC) class 1.
NK cells (belonging to the group of innate lymphoid cells) are defined as large granular lymphocytes (LGL) and constitute the third kind of cells differentiated from the common lymphoid progenitor generating B and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow (BM), lymph node, spleen, tonsils and thymus where they then enter into the circulation. NK cells differ from Natural Killer T cells (NKT) phenotypically, by origin and by respective effector functions; often NKT cell activity promotes NK cell activity by secreting IFNγ. In contrast to NKT cells, NK cells do not express T-cell antigen receptors (TCR) or Pan T marker CD3 or surface immunoglobulins (Ig) B cell receptors, but they usually express the surface markers CD16 (FcγRIII) and CD56 in humans, NK1.1 or NK1.2 in C57BL/6 mice. Up to 80% of human NK cells also express CD8.
In addition to the knowledge that natural killer cells are effectors of innate immunity, recent research has uncovered information on both activating and inhibitory NK cell receptors which play important function roles including self tolerance and sustaining NK cell activity. NK cell also play a role in adaptive immune response, numerous experiments have worked to demonstrate their ability to readily adjust to the immediate environment and formulate antigen-specific immunological memory, fundamental for responding to secondary infections with the same antigen. The ability for NK cells to act in both the innate and adaptive immune response is becoming increasingly important in research utilizing NK cell activity and potential cancer therapies.
NK cell receptors can also be differentiated based on function. Natural cytotoxicity receptors directly induce apoptosis after binding to ligands that directly indicate infection of a cell. The MHC dependent receptors (described above) use an alternate pathway to induce apoptosis in infected cells. Natural killer cell activation is determined by the balance of inhibitory and activating receptor stimulation i.e. if the inhibitory receptor signaling is more prominent then NK cell activity will be inhibited, similarly if the activating signal is dominant then NK cell activation will result.
Activating receptors: Ly49 (homodimers)—a relatively ancient, C-type lectin family receptor; are of multigenic presence in mice, while humans have only one pseudogenic Ly49; the receptor for classical (polymorphic) MHC I molecules. NCR (natural cytotoxicity receptors), upon stimulation, mediate NK killing and release of IFNY. CD94:NKG2 (heterodimers)—a C-type lectin family receptor, conserved in both rodents and primates and identifies non-classical (also non-polymorphic) MHC I molecules like HLA-E. Expression of HLA-E at the cell surface is dependent on the presence of nonamer peptide epitope derived from the signal sequence of classical MHC class 1 molecules, which is generated by the sequential action of signal peptide peptidase and the proteasome. Though indirect, this is a way to survey the levels of classical (polymorphic) HLA molecules. CD16 (FcγIIIA) play a role in antibody-dependent cell-mediated cytotoxicity (ADCC), in particular they bind IgG.
Inhibitors Receptors: KIR (Killer-cell immuoglobulin-like receptors)—belong to a multigene family of more recently-evolved Ig-like extracellular domain receptors; are present in non-human primates; and are the main receptors for both classical MHC 1 (HLA-A, HLA-B, HLA-C) and also non-classical HLA-G in primates. Some KIRs are specific for certain HLA subtypes. Most KIR are inhibitory and dominant. Regular cells express MHC class 1 and therefore are recognized by KIR receptors and NK cell killing is inhibited. ILT or LIR (leukocyte inhibitory receptors)—are recently-discovered members of the Ig receptor family. Ly49 (homodimers)—a C-type lectin family of receptors. Are of multigenic presence in mice, while humans have only one pseudogenic Ly49. Both activating and inhibitory isoforms exist. Highly polymorphic on the population level. Even though they are structurally unrelated to KIR:s, they are the functional homologues of KIR:s in mice, including the expression pattern, Ly49:s are receptor for classical (polymorphic) MHC I molecules.
NK cell function: Cytolytic granule mediated cell apoptosis: NK cells are cytotoxic; small granules in their cytoplasm contain proteins such as perforin and proteases known as granzymes. Upon release in close proximity to a cell slated for killing, perforin forms pores in the cell membrane of the target cell, creating an aqueous channel through which the granzymes and associated molecules can enter, inducing either apoptosis or osmotic cell lysis. The distinction between apoptosis and cell lysis is important in immunology: lysing a virus-infected cell could potentially only release the virions, whereas apoptosis leads to destruction of the virus inside, α-defensins, an antimicrobial is also secreted by NK cells, directly kills bacteria by disrupting their cell walls analogous to neutrophils.
Antibody-dependent cell-mediated cytotoxicity (ADCC): Infected cells are routinely opsonized with antibodies for detection by immune cells. Antibodies that bind to antigens can be recognized by FcγRIII (CD16) receptors expressed on NK cells resulting in NK activation, release of cytolytic granules and consequent cell apoptosis. This is a major mechanism of killing for some monoclonal antibodies like rituximab (Rituxan), ofatumumab (Azzera) and others.
Cytokine-induced NK and CTL activation: Cytokines play a crucial role in NK cell activation. As these are stress molecules released by cells upon viral infection, they serve to signal to the NK cell the presence of viral pathogens. Cytokines involved in NK activation include IL-12, IL-15, IL-18, IL-2, and CCL5. NK cells are activated in response to interferons or macrophage-derived cytokines. They serve to contain viral infections while the adaptive immune response is generating antigen-specific cytotoxic T cells that can clear the infection. NK cells work to control viral infections by secreting IFNγ and TNFα. IFNγ activates macrophages for phagocytosis and lysis, and TNFα acts to promote direct NK tumor cell killing. Patients deficient in NK cells prove to be highly susceptible to early phases of herpes virus infection.
Tumor cell surveillance: Natural killer cells often lack antigen-specific cell surface receptors and therefore are part of innate immunity, i.e. able to react immediately with no prior exposure to the pathogen. In both mice and humans, NKs can be seen to play a role in tumor immuno-surveillance by directly inducing the death of tumor cells (NKs act as cytolytic effector lymphocytes), even with the absence of surface adhesion molecules and antigenic peptides. This role of NK cells is critical for immune success particularly because T cells are unable to recognize pathogens in the absence of surface antigens. Tumor cell detection results in activation of NK cells and consequent cytokine production and release.
If the tumor cells do not cause inflammation, they will also be regarded as self and therefore will not induce a T cell response. A number of cytokines are produced by NKs, including tumor necrosis factor α (TNFα), IFNγ, and interleukin (IL-10). TNFα and IL-10 act as pro-inflammatory and immuno-suppressors, respectively. The activation of NK cells and subsequent production of cytolytic effector cells impacts macrophages, dendritic cells, and neutrophils, which subsequently affects antigen-specific T and B cell responses. Instead of acting via antigen-specific receptors, lysis of tumor cells by NK cells is mediated by alternative receptors, including NKG2D, NKp44, NKp46, NKp30, and DNAM. NKG2D is a disulfide-linked homodimer which recognizes a number of ligands, including ULBP and MICA, which are typically expressed on tumor cells.
NK cells, along wall macrophages and several other cell types, express the Fc receptor (FcR) molecule (FC-gamma-RIII=CD16), an activating biochemical receptor that binds the Fc portion of antibodies. This allows NK cells to target cells against which a humoral response has been mobilized and to lyse cells through antibody-dependent cellular cytotoxicity (ADCC). To determine the ADCC contribution of monoclonal antibodies, NK-92 cells (a “pure” NK cell line) has been transacted with the gene for the high-affinity FcR.
Anti-cancer therapies using expanded NK cells: NK cells are currently being used in multiple clinical trials to treat several different types of cancer. Current protocols include treating cancer patients with ex vivo IL-2 expanded NK cells and treating patients with the monoclonal antiKIR antibody, 1-7F9, designed to block NK cell inhibitory receptors. Because of the limited number of NK cells in blood (only 10% of lymphocytes are NK-cells) their number needs to be expanded in culture. This can take a few weeks and the yield is donor dependent. Treating large numbers of patients with IL-2 expanded NK cells is expensive and may not be feasible to do on a large scale. Treating patients with 1-7F9 mAb, by design, may not necessarily result in sustained NK cell activation.
There is a need to develop new methods of inducing broad activation of NK cells to treat or prevent various diseases or conditions.
The present invention is directed to overcoming these and other deficiencies in the art.